The mechanisms by which migrating leukocytes penetrate the subendothelial basement membrane are poorly understood. We previously showed that prolonged culture of human umbilical vein endothelial cells (HUVEC) was associated with the deposition of a distinct basement membrane which modified neutrophil recruitment induced by inflammatory cytokines (Butler et al., 2005). Here, we modified this in vitro model to study the migration of neutrophils through the underlying basement membrane as well as the endothelial cell monolayer. Confluent first-passage HUVEC were cultured for 4 or 20 days on 3µm pore transwell inserts and then treated with 0, 1 or 100U/ml tumour necrosis factor α (TNFα) or 50pg/ml interleukin-1β for 4 hours. Purified human neutrophils were added to the surface and their migration was assessed after 0.5, 1, 2 or 24h by collecting and counting neutrophils from the upper and lower chambers, and by fixing and staining the filters to quantify neutrophil number and location above and below the filter. Neutrophil migration through the HUVEC and basement membrane increased with dose of cytokine and with time, but was consistently delayed for 20-day cultures compared to 4-day cultures. For example, 34.5 ± 3.0% of neutrophils added migrated through 4-day HUVEC treated with 100U/ml TNFα after 0.5h, compared to only 14.5 ± 5.9% migrated through 20-day HUVEC (mean ± SEM from 8 experiments; P=85%. Interestingly, antibody-blockade of platelet-endothelial cell adhesion molecule-1 (CD31) on neutrophils and endothelial cells had no effect on migration induced by either cytokine, at 4 or 20 days. We have shown that the basement membrane produced by human endothelial cells delays neutrophil migration. Transmigration required β2-integrin engagement, but this could not be associated specifically with migration through the basement membrane. The model developed allows the further study of the molecular mechanisms by which neutrophils cross the distinct barriers presented by endothelial cells and their basement membrane.